Stem cells represent a major resource for therapeutic approaches to treat disease and for regenerative medicine. However, a major barrier in the use of stem cells for widespread application is the lack of understanding of specific cell characteristics and the inability to isolate pure suspensions of these cells. Production of sperm depends on the coordinated homeostasis of the germline stem cell, also called spermatogonial stem cells (SSCs). Like all adult stem cell populations, dysregulation of SSCs results in compromised tissue function, specifically the lack of sperm production. In addition, the parameters of SSC formation during fetal and postnatal development suggests that lack of control of SSC activity may lead to testicular cancer, the most prevalent cancer in men aged 15 to 34. Greater than 90% of testicular cancers are germ cell derived. Thus, a better understanding of SSCs, their location in the tissue and their interactions with other cells are of great importance to understanding and solving diseases associated with SSC dysregulation and will provide insight into the function of other adult stem cells. A barrier to understanding stem cell function is our inability to isolate and characterize pure stem cells. Our approach takes advantage of two novel and state of the art techniques to investigate stem cells. First, the GFP label retention by stem cells in a bitransgenic mouse model following doxycycline induced expression of the H2B-GFP fusion protein. After the doxycycline-induced pulse is removed, a chase period results in the loss of GFP label in all cells except slowly dividing stem cells. Second, we will transplant label- retaining cells to determine their stem cell properties. Thus this innovative approach with existing mouse models provides the methodology to test the hypothesis that long-term label retaining cells in the testis are SSCs and we can isolate and characterize these cells. We will test this hypothesis by transplantation of label retaining cells to evaluate stem cell function and gene expression profiling of label retaining cells to characterize their stem cell properties. Completion of the research described here has the potential to provide valuable insights about stem cell biology and provide new methodologies for the investigation and application of stem cells. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because the discovery of stem cell characteristics and their interactions in tissues will ultimately provide new methodologies to treat human disease and for regenerative medicine. Thus the proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will help reduce human disease.